Bacterial cultures are widely produced for inclusion in products such as forage inoculants, probiotics and fermented foods. Cultures are typically prepared by fermentation; they are grown in large volumes of enrichment broth, either by shaken-flask, solid-state or continuous fermentation. Once the desired cell population is reached, the cells are harvested from the production fermentor and preserved by cryopreservation and/or lyophilization. See e.g. Manual of Industrial Microbiology and Biotechnology, ASM, Washington, D.C., Demain, A. L., Solomon, N. A. (eds.) (1986). After preservation, cultures may be blended into commercial product and stored.
At each step, from fermentation to commercial product preparation, the cells are subject to constantly changing environments which lead to various types of stress and injury. Typical stresses include pH fluctuations, depletion of essential nutrients and accumulation of metabolic by-products. Concentration and freezing of cells after growth can constitute additional stress.
Freezing often produces cold shock and leads to the formation of intracellular ice. Freeze-drying is typically conducted by sublimation of water. Freeze-dried cultures are stored under refrigeration or frozen in dry, moisture-proof packaging until inclusion in commercial products. When cultures are used in commercial formulations, cells are further insulted due to mechanical injury and long term storage.
The stress on bacterial cultures, from fermentation through commercial product inclusion, lead to cell death and injury. Loss of viable cells due to the above stress results in loss of active product to the end user. Because of decreased viability, the product may not have desired efficacy or meet guaranteed specifications; therefore additional culture is typically included in the commercial product to assure adequate performance. If cultures used to prepare products contain stressed and injured cells, the product may not have stability to withstand the additional stress of long-term storage. Thus, product efficacy may decrease over time. Drop in culture viability results in additional expense to the manufacturer due to product recall or fortification to meet label specifications.
To prevent manufacture of bacterial products with stressed culture, it is necessary to screen cultures for viability and vitality prior to inclusion into a commercial finished product. The usual method for detecting microorganisms is by the conventional plate count method as described by the FDA Bacteriological Analytical Method, Washington, D.C.: AOAC, (1984). According to this method, viable microbial cells are placed onto a solid medium, containing all the nutrients essential for growth, and the inoculated medium is incubated under conditions favorable for growth. The cells reproduce on the medium to form visible colonies that comprise cloned generations of the original cell. See Microbial Ecology: Principles, Methods and Applications, Levin, M. A., Seidler, R. J., Rogul, M. (eds), McGraw Hill, Inc., New York, (1992). This method, limited to assessing only those cells which are live, uninjured or capable of recovery on the standard microbial medium, typically requires several days of incubation.
Current practices to determine culture suitability for product inclusion are performed by long-term shelf-life stability studies. This method requires storage of culture under different environmental conditions for up to twelve months. Culture viability counts are verified during the time period in storage by the conventional plate count method described above. This standard procedure requires a long time interval for stability testing during which it is not possible to predict the population of cells most likely to die. Unless the injured cells are recoverable on standard agar, they are not included in the viable population.
Based on the foregoing, there exists a need to provide assay methods to predict the viability of bacterial cells that allow for rapid determination of live, dead and/or stressed cells in a culture. There exists a further need to rapidly evaluate the relative health of a population of cells.
It is therefore an object of the present invention to provide methods of rapidly determining the proportion of live, dead or stressed cells in a culture.
It is a further object of the present invention to provide a rapid quantitative indicator of the relative health of a given population of cells.
It is a further object of the present invention to provide methods of predicting the long-term stability of a given culture.
It is a further object of the present invention to provide means of meeting specifications of a given culture at minimal expense.
These and other objects of the present invention will become readily apparent from the ensuing description.